Project summary Extracellular adenosine triphosphate (ATP) is an evolutionary conserved ?danger signal? used to sense cellular damage. In mice and humans, extracellular ATP (eATP) is recognized by purinergic receptors. Among those receptors, P2RX7 is particularly relevant since it is preferentially expressed in immune cells, being able to activate both innate and adaptive immune responses. Functional adaptive immune responses ? especially T cell memory ? are crucial for the control of viral infections. Notably, our recently published work shows that P2RX7 is crucial for the generation and maintenance of virus-specific long-lived memory CD8+ T cells. P2RX7 seems to control the metabolic fitness ? in particular, mitochondrial function and viability ? of CD8+ T cells throughout the immune response. Although these findings indicate that P2RX7 is required at different time points during an effector immune response, it is not clear when during an immune response P2RX7/eATP signaling is required for the full development of memory CD8+ T cells. Another unexplored question lies on the source of eATP for P2RX7 activation in CD8+ T cells throughout the immune response. eATP can originate from the surrounding microenvironment (being CD8+ T cell-extrinsic), which is likely to happen during primary or secondary antigen responses, or can be released by the own CD8+ T cell via Pannexin 1 (Panx1) channels (being CD8+ T cell- intrinsic) ? this can be particularly relevant during long-term memory, when inflammation recedes. In Aim 1, we will use P2RX7 and Panx1 ablation tools to elucidate whether P2RX7/eATP sensing is required throughout the effector phase or only during early activation ? as well as the source of eATP for this stage. It is also unclear whether sustained P2RX7/eATP signaling is required for long-term survival of memory CD8+ T cells. In Aim 2 we will use tools to knockout P2RX7 and Panx1 after memory establishment to answer this question and to elucidate the eATP source potentially involved in long-term survival maintenance. Finally, in Aim 3, we will evaluate if the recall responses of memory CD8+ T cells in response to a secondary antigen encounter (and the subsequent protective immune response) requires P2RX7-mediated eATP signaling. These studies will help elucidate the role of eATP sensing for memory CD8+ T cell homeostasis and will provide key insights on how ?danger signals? and the sensing of cellular damage help build not only short-term but also long-term immunity against pathogens. This will provide the basis for the improvement of future vaccine and antiviral immunotherapy designs.